Considerable evidence exists for membrane skeletal derangements, as well as changes in calcium metabolism and protein phosphorylation, in the sickle erythrocyte. The goal of this application is to gain a better understanding of the role of calcium and protein phosphorylation in 1 erythrocyte function, particularly as they affect the membrane skeleton. The research will focus on a recently-identified calcium-dependent protein phosphorylation system in erythrocytes, that of protein kinase C (PK-C) and its membrane-associated substrates. The functions of PK-C will be probed by treating erythrocytes with phorbol esters which enter the cell and activate PK-C. These compounds also cause PK-C to rapidly translocate to the membrane where the enzyme phosphorylates a number of membrane skeletal substrates. Long-term treatment of red cells with phorbol esters leads to a loss of PK- C activity. The biochemical basis for this phenomenon will be investigated. PK-C will be purified from red cells and its properties compared to that of the brain enzyme. The basis for other effects of calcium on protein phosphorylation will be determined and the role of the calcium regulatory protein, calmodulin, in mediating these processes will be established. A pair of membrane skeletal substrates for PK-C, collectively termed adducin, will then be studied in detail. We have already purified these proteins from the erythrocyte membrane, examined their physicochemical and some of their biochemical properties, and prepared antibodies to them. Studies from this and other laboratories suggest that these proteins may play an important role in regulating membrane skeletal behavior. However, the precise function of adducin in the skeleton is still unsettled. We will investigate the interaction of adducin with other skeletal proteins (primarily actin and spectrin) in purified systems where the individual elements are recombined. In addition we will assess the possibility that adducin interacts directly with the inside face of the membrane and identify the membrane component responsible for this binding. The effects of calmodulin and phosphorylation on each of these interactions will be investigated. The composition of adducin-actin-(spectrin) complexes will be examined by electron microscopy. The localization of adducin in the intact erythrocyte membrane skeleton will be studied by immunoelectron microscopy and the locali of adducin in other types of cell (e.g. fibroblasts) by immunofluorescence. The biogenesis of adducin in the developing erythrocyte will be studied in erythropoietin-stimulated friend virus-infected mouse erythroblasts, and its role in the assembly of the mature membrane skeleton determined. It is hoped that these investigations will throw light on the regulation of protein interactions in the erythrocyte membrane and will contribute to our understanding of pathological states.